Fig. 3: F-actin barbed ends and F-actin pointed ends are present at stereocilia tips.

a EGFP-TMOD1 transfected P5 IHCs, either unextracted or extracted by saponin before fixation, that were also stained with phalloidin for F-actin (magenta). b Representative line scans drawn down the center of stereocilia of EGFP-TMOD1 transfected, unextracted P5 IHCs. The peak EGFP level was set as 0 on x axis and the fluorescence intensity was normalized to the maximal fluorescence intensity of row 1. c Line scans quantifying EGFP-TMOD1 levels from stereocilia tips toward shafts in saponin-extracted or unextracted P5 IHCs. Sample size (stereocilia, cells, cochleae): unextracted (18, 6, 2), extracted (33, 11, 3). The shadow lines represent individual stereocilia; the thick solid lines are the average level of all stereocilia. The data were plotted as mean ± SD and analyzed by two-way ANOVA (P < 0.0001 for the effects of extraction, distance from stereocilia tips, and interactions between these parameters, based on stereocilia). d His-TMOD1, DNaseI, or His-CAPZ (green, grey) localization after probing permeabilized IHCs at P5-6. F-actin was stained with phalloidin (magenta). The experiments were repeated three times with similar results. e Diagrams of potential actin structures in stereocilia with F-actin barbed and pointed ends bound by His-CAPZ and His-TMOD1, respectively. f Lattice SIM images of His-TMOD1, DNaseI, and His-CAPZ (cyan, thermal lookup table) at row 1 stereocilia tips with phalloidin-stained F-actin (magenta), which were quantified in (g, h). Magenta arrowheads denote the position of peak fluorescence intensity. g Representative line scans drawn down the center of stereocilia showing the intensity of His-TMOD1 (blue), DNaseI (purple), His-CAPZ (red), and F-actin actin (black). The stereocilia tip is indicated by black dashed arrow. Peak intensities for His-TMOD1, DNaseI, and His-CAPZ are indicated by colored dashed arrows. The offsets of the probe centers from the stereocilia tips were determined and plotted in (h). h A frequency histogram showing the pixel offsets of His-TMOD1 (blue), DNaseI (purple), and His-CAPZ (red) from the stereocilia tip. Sample size (stereocilia, cells, cochleae) of each probe (75, 15, 3). The histogram of each probe is fitted with a Gaussian curve. Mean offsets for peak of the Gaussian curves based on the pixel size (31 nm x 31 nm): His-TMOD1, 16 nm; DNaseI, 24 nm; His-CAPZ, 51 nm. R-squared value of the fit: His-TMOD1, 0.996; DNaseI, 0.976; His-CAPZ, 0.982. Scale bars represent 5 μm. Source data are provided as a Source Data file.